Next-generation sequencing (NGS) is now widely used in cancer research and is also becoming an important clinical tool. Using targeted libraries, NGS can interrogate specific genome regions at extremely high depth and therefore identify rare mutations even in relatively heterogeneous clinical samples such as cytology samples or bodily fluids.
However, mutant detection levels are still limited by the error rate inherent in current NGS protocols, such that reliable detection of mutant fractions below 1-2% remains challenging. While this is adequate for many applications, there are a number of very exciting avenues of cancer research that require even more sensitive approaches. Perhaps the best example of this is the detection of mutations in bodily fluids such as plasma, urine, sputum and others where DNA from a few cancer cells is present in a vast excess of normal cell DNA. Applications such as cancer diagnosis, monitoring response to therapy and monitoring the evolution of tumor heterogeneity via liquid biopsy (plasma) all require detection of mutations at frequencies below 0.1%. Recently developed experimental methods combined with new algorithms for variant calling are capable of achieving this sensitivity but require large amounts of relatively good quality DNA, or can only analyze very limited genomic regions (single PCR amplicons).